ACE2 Peptide Fragment Interaction with Different S1 Protein Sites.

Unlabelled: We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry.

Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.

Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule.

Computational simulation of ligand docking to L-type pyruvate kinase subunit.

Computational blind docking approach was used for mapping of possible binding sites in L-type pyruvate kinase subunit for peptides, RRASVA and the phosphorylated derivative RRAS(Pi)VA, which model the phosphorylatable N-terminal regulatory domain of the enzyme. In parallel, the same docking analysis was done for both substrates of this enzyme, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), and for docking of fructose 1,6-bisphosphate (FBP), which is the allosteric activator of the enzyme. The binding properties of the entire surface of the protein were scanned and several possible binding sites were identified in domains A and C of the protein, while domain B revealed no docking sites for peptides or for substrates or the allosteric regulator.

Computer modeling of the dynamic properties of the cAMP-dependent protein kinase catalytic subunit.

The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the "opened" and "closed" structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme-ATP and enzyme-PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands.

Kinetic sonication effects in aqueous acetonitrile solutions. Reaction rate levelling by ultrasound.

The kinetics of the pH-independent hydrolysis of 4-methoxyphenyl dichloroacetate were investigated with and without ultrasonic irradiation in acetonitrile-water binary mixtures containing 0.008 to 35 wt.% of acetonitrile and the kinetic sonication effects (kson/knon) were calculated.

Kinetic sonication effects in light of molecular dynamics simulation of the reaction medium.

Molecular dynamics (MD) simulation of the structure of ethyl acetate solutions in two water-ethanol mixtures was performed at 280 and 330K. The MD simulations revealed that ethyl acetate was preferentially solvated by ethanol, water being mainly located in the next solvation layer. With increasing temperature ethanol was gradually replaced by water in the first solvation shell.

Nβ-methylation changes the recognition pattern of aza-β3-amino acid containing peptidomimetic substrates by protein kinase A.

The protein kinase A (PKA)-catalyzed phosphorylation of peptide substrate RRASVA analogs, containing Nβ-Me-aza-β3-amino acid residues in all subsequent positions, was studied. This work follows along the lines of our previous research of the phosphorylation of aza-β3-analogs of RRASVA (the shortest active substrate of PKA) and allows characterizing the influence of Nβ-methylation of aza-β3-amino acid residues on substrate recognition by PKA on substrate binding and phosphorylation steps. It was found that the effect of Nβ-methylation was dependent upon the position of the structure alteration.

Effect of two simultaneous aza-β3-amino acid substitutions on recognition of peptide substrates by cAMP dependent protein kinase catalytic subunit.

Peptidomimetic analogs of the hexapeptide RRASVA, containing simultaneously two aza-β(3)-amino acid residues in different positions of this sequence, except for the phosphorylatable serine residue, were synthesized and tested as substrates for the cAMP-dependent protein kinase catalytic subunit. All these peptidomimetics were phosphorylated by the enzyme and this reaction was characterized by the K(m) and k(cat) values as well as by the second-order rate constants k(II). Affinity and reactivity of all peptidomimetics was lower than that for the parent peptide RRASVA.

Aza-beta(3)-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates.

Peptidomimetic analogs of the peptide RRASVA, known as the "minimal substrate" of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-beta(3) analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates.

Mechanism and stoichiometry of 2,2-diphenyl-1-picrylhydrazyl radical scavenging by glutathione and its novel alpha-glutamyl derivative.

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing alpha-glutamyl residue in place of the gamma-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.

Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6.

Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.